Macrocyclic compounds and methods of treatment

ABSTRACT

The instant invention describes macrocyclic compounds having therapeutic activity, and methods of treating disorders such as cancer, tumors and cell proliferation related disorders, or affect cell differentiation, dedifferentiation or transdifferentiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplications Nos. 60/970,986, filed 9 Sep. 2007; 61/009,903, filed 3Jan. 2008; 61/030,993, filed 24 Feb. 2008; 61/125,542, filed 24 Apr.2008 and a Provisional Patent Application 61/189,093 filed 15 Aug. 2008which has not yet been assigned an application number, the entireteachings of which are hereby incorporated by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by a NOAA, Office of Sea Grant, U.S.Department of Commerce Grant No. NA060AR4170014. The government hascertain rights in the invention.

BACKGROUND

The identification of new pharmacophores is of paramount biomedicalimportance and natural products have recently been regaining attentionfor this endeavor.¹ This renaissance is closely tied to the successfulexploitation of the marine environment which harbors unmatchedbiodiversity that is presumably concomitant with chemical diversity.² Inparticular, marine cyanobacteria are prolific producers of bioactivesecondary metabolites,³ many of which are modified peptides orpeptide—polyketide hybrids with promising antitumor activities, such asdolastatin 10,⁴ curacin A,⁵ and apratoxin A.⁶ As a result of ongoinginvestigations to identify new drug leads from cyanobacteria in Florida,we report here the structure determination and preliminary biologicalcharacterization of a marine cyanobacterial metabolite with novelchemical scaffold and nanomolar antiproliferative activity. Thesefindings provide new alternatives to address unmet needs in thetreatment of proliferation diseases and disorders. The compounds hereinare also now found to mediate histone deacetylase (HDAC) processes(e.g., inhibition) and as such are useful for treating diseases,disorders, or symptoms thereof mediated by inhibition of histonedeacetylase (HDAC). These findings provide new alternatives to addressunmet needs in the treatment of HDAC mediated diseases and disorders.

BRIEF SUMMARY OF THE INVENTION

The invention is directed towards macrocyclic compounds, and methods oftreating disease and disorders, including proliferation diseases anddisorders, and HDAC mediated diseases and disorders, by use of thecompounds and compositions thereof.

The invention is directed towards macrocyclic compounds, methods ofmodulating proliferation activity, and methods of treating proliferationdisease and disorders.

In one embodiment, the invention provides a compound according toFormula I:

wherein:

each R is independently H or optionally substituted alkyl;

each R¹ is independently H or optionally substituted alkyl;

each R² is independently H, optionally substituted alkyl, or C(O)R;

each R³ is independently H, optionally substituted alkyl, C(O)OR, orC(O)NRR;

each R⁴ is independently H, optionally substituted alkyl, C(O)OR, orC(O)NRR;

and pharmaceutically acceptable salts, solvates, or hydrates thereof.

Another aspect is a compound of formula Ia (and pharmaceuticallyacceptable salts, solvates, or hydrates thereof), where R, R¹, R², R³,and R⁴ are as defined in formula I:

Other embodiments include a compound of any of the formulae herein,wherein R³ and R⁴ are H; wherein R¹ is isopropyl; wherein R² is alkyl;wherein R² is alkylC(O)—; wherein R² is H; wherein the compound is anyof Compounds 1-8 in Table A; or wherein the compound is largazole.

In certain instances, the compounds of the invention are selected fromthe following of Formula (I) (including formula Ia) having thestructure:

TABLE A Cmpd No. R¹ R² R³ R⁴ 1 isopropyl n-heptylC(O)— H H 2 isopropyln-heptylC(O)— H Me 3 isopropyl Me H H 4 isopropyl n-heptylC(O)— HmethylC(O)— 5 isopentyl n-heptylC(O)— H H 6 ethyl n-heptylC(O)— Me Me 7isopropyl CH₃C(O)— H H 8 isopropyl H H H

In another aspect, the invention provides a pharmaceutical compositioncomprising the compound of formula I and a pharmaceutically acceptablecarrier.

In other aspects, the invention provides a method of treating a disease,disorder, or symptom thereof in a subject, comprising administering tothe subject a compound of any of the formulae herein (e.g., formula I,formula Ia). In another aspect, the compound is administered in anamount and under conditions sufficient to ameliorate the disease,disorder, or symptom thereof in a subject.

In other aspects, the invention provides a method of modulating HDACactivity in a subject, comprising contacting the subject with a compoundof any of the formulae herein (e.g., formula I, formula Ia), in anamount and under conditions sufficient to modulate HDAC activity. Inanother aspect, the modulation is inhibition.

In other aspects, the invention provides a method of modulating theproliferation activity in a subject, comprising contacting the subjectwith a compound of formula I, in an amount and under conditionssufficient to modulate proliferation activity.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a proliferation related disorder ordisease, comprising administering to the subject an effective amount ofa compound or pharmaceutical composition of formula I.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a proliferation related activityrelated disorder or disease, wherein the subject has been identified asin need of treatment for a proliferation related disorder or disease,comprising administering to said subject in need thereof, an effectiveamount of a compound or pharmaceutical composition of formula I, suchthat said subject is treated for said disorder.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a cell proliferation related disorderor disease, wherein the subject has been identified as in need oftreatment for a cell proliferation related disorder or disease,comprising administering to said subject in need thereof, an effectiveamount of a compound or pharmaceutical composition of formula I, suchthat cell proliferation in said subject is modulated (e.g., downregulated). In another aspect, the compounds delineated hereinpreferentially target cancer cells over nontransformed cells.

In a specific aspect, the invention provides a method of treatingcancer, tumor growth, cancer of the colon, breast, bone, brain andothers (e.g., osteosarcoma, neuroblastoma, colon adenocarcinoma),comprising administering to said subject in need thereof, an effectiveamount of a compound delineated herein (e.g., Formula I), andpharmaceutically acceptable salts thereof. Other cancers that may betreated by the compositions and methods of the invention include cardiaccancer (e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma andteratoma); lung cancer (e.g., bronchogenic carcinoma, alveolarcarcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma); various gastrointestinal cancer (e.g., cancersof esophagus, stomach, pancreas, small bowel, and large bowel);genitourinary tract cancer (e.g., kidney, bladder and urethra, prostate,testis; liver cancer (e.g., hepatoma, cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bonecancer (e.g., osteogenic sarcoma, fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma,cutaneous T-cell lymphoma, multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma, benign chondroma, chondroblastoma,chondromyxofibroma, osteoid osteoma and giant cell tumors); cancers ofthe nervous system (e.g., of the skull, meninges, brain, and spinalcord); gynecological cancers (e.g., uterus, cervix, ovaries, vulva,vagina); hematologic cancer (e.g., cancers relating to blood, Hodgkin'sdisease, non-Hodgkin's lymphoma); skin cancer (e.g., malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis);and cancers of the adrenal glands (e.g., neuroblastoma). Other diseasesand disorders that can be treated include the treatment of inflammatorydisorders, neurodegenerative diseases, protozoal and latent viralinfections, and (fibro)proliferative disorders.

In another aspect, the invention provides a method of inhibiting histonedeacetylase (HDAC) in a subject in need thereof comprising administeringto said subject, an effective amount of a compound delineated herein(e.g., Formula I), and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides a method of treating diseases,disorders, or symptoms thereof mediated by inhibition of histonedeacetylase (HDAC) in a subject in need thereof comprising administeringto said subject, an effective amount of a compound delineated herein(e.g., Formula I), and pharmaceutically acceptable salts thereof.Recently, HDAC inhibitors have been found to ameliorate progression ofthe spinal muscular atrophy (SMA) motor neuron disease and theHuntington disease mouse models. The neuroprotective role of HDACinhibitors seems to extend to other diseases that share mechanisms ofoxidative stress, inflammation and neuronal cell apoptosis. HDACinhibitors also have widespread modulatory effects on gene expressionwithin the immune system and have been used successfully in the lupusand rheumatoid arthritis autoimmune disease models. Recently, theefficacy of the HDAC inhibitor Trichostatin A was established inameliorating disease in the multiple sclerosis (MS) animal model,experimental autoimmune encephalomyelitis (EAE). In aspects, thecompounds herein are useful to treat MS, an autoimmune, demyelinatingand degenerative disease of the human central nervous system (CNS). Inaspects the compounds herein are useful to treat stroke. In otheraspects, the HDAC inhibitor compounds are useful to treat or preventmemory loss, for inducing neurogenesis, for enhancing memory retention,for enhancing memory formation, for increasing synaptic potential ortransmission, or for increasing long term potentiation (LTP). Histonedeacetylases (HDAC) are also associated with a variety oftranscriptional repressors that control cellular differentiation andproliferation. Modulation of gene expression through HDAC inhibition maycontrol stem cell fate and affect differentiation, dedifferentiation ortransdifferentiation. Thus, the compounds herein are useful to improvereprogramming efficiency; enable efficient induction of pluripotent stemcells; cause pluripotent stem cells to cease proliferating and enterterminal differentiation pathways; to inhibit differentiation tooligodendrocytes, where HDAC2 activity specifically inhibitsdifferentiation to astrocytes, while HDAC1 activity is required fordifferentiation to neurons; enhance differentiation in stem celltherapy, be used as medium supplements that stabilize the phenotype ofprimary cells in culture; stimulate osteoblast maturation; implementbone tissue engineering; induce myogenic differentiation; up-regulatbasal activity of transcription from a MyoD-responsive reporter; andinduce ex vivo expansion of human hematopoietic stem cells (HSC). Inaspects the compounds herein are useful to treat conditions in a subjectincluding but not limited to myogenesis, neurogenesis, osteogenesis andosteoblast maturation.

In another aspect, the invention provides a method of treating diseases,disorders, or symptoms in a subject in need thereof comprisingadministering to said subject, an effective amount of a compounddelineated herein (e.g., Formula I), and pharmaceutically acceptablesalts thereof. Such methods are useful for treating memory loss,inducing neurogenesis, enhancing memory retention, enhancing memoryformation, increasing synaptic potential or transmission, or increasinglong term potentiation (LTP). Such methods are also useful for treatingdiseases and disorders associated with stem cell fate and that areaffected by differentiation, dedifferentiation or transdifferentiation,and thus include but not limited to myogenesis, neurogenesis,osteogenesis and osteoblast maturation.

In another aspect, the compounds of any of the formulae herein (e.g.,formula (I)) are compounds having class I HDAC selectivity, thus theyare useful as anticancer agents; and furthermore having selectivity forclass I HDAC versus class II HDAC also provides a more desirabletherapeutic profile as it is indicated that inhibition of certainspecific class II HDACs may have undesirable consequences, including forexample, promoting cardiac hypertrophy. See, Furumai et al. CancerResearch 2002, 62, 4916-4921; Yurek-George et al. J. Med. Chem. 2007,50, 5720-5726. Thus, in one aspect, the compounds and methods herein arethose wherein the compounds demonstrate selectivity in class I/class IIHDAC selectivity (e.g., at least 2-fold, at least 10-fold, at least100-fold, at least 1000-fold, at least X-fold where X is any numberbetween 1 and 100,000 inclusive).

Methods delineated herein include those wherein the subject isidentified as in need of a particular stated treatment. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to thefollowing non-limiting examples and with reference to the followingfigures, in which:

FIG. 1. depicts largazole dissociation pathways and structures ofproduct ions.

FIG. 2. depicts HDAC inhibition results.

FIGS. 3-9 are NMR spectra for largazole.

DETAILED DESCRIPTION Definitions

In order that the invention may be more readily understood, certainterms are first defined here for convenience.

As used herein, the term “treating” a disorder encompasses preventing,ameliorating, mitigating and/or managing the disorder and/or conditionsthat may cause the disorder. The terms “treating” and “treatment” referto a method of alleviating or abating a disease and/or its attendantsymptoms. In accordance with the present invention “treating” includespreventing, blocking, inhibiting, attenuating, protecting against,modulating, reversing the effects of and reducing the occurrence ofe.g., the harmful effects of a disorder.

As used herein, “inhibiting” encompasses preventing, reducing andhalting progression.

The term “modulate” refers to increases or decreases in the activity ofa cell in response to exposure to a compound of the invention.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. Particularly, in embodiments thecompound is at least 85% pure, more preferably at least 90% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

A “peptide” is a sequence of at least two amino acids. Peptides canconsist of short as well as long amino acid sequences, includingproteins.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “protein” refers to series of amino acid residues connected oneto the other by peptide bonds between the alpha-amino and carboxy groupsof adjacent residues.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3rd ed., 1994) and Cantor and Schimmel, BiophysicalChemistry Part I. The Conformation of Biological Macromolecules (1980).“Primary structure” refers to the amino acid sequence of a particularpeptide. “Secondary structure” refers to locally ordered, threedimensional structures within a polypeptide. These structures arecommonly known as domains. Domains are portions of a polypeptide thatform a compact unit of the polypeptide and are typically 50 to 350 aminoacids long. Typical domains are made up of sections of lesserorganization such as stretches of β-sheet and α-helices. “Tertiarystructure” refers to the complete three dimensional structure of apolypeptide monomer. “Quaternary structure” refers to the threedimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

The term “administration” or “administering” includes routes ofintroducing the compound(s) to a subject to perform their intendedfunction. Examples of routes of administration which can be used includeinjection (subcutaneous, intravenous, parenterally, intraperitoneally,intrathecal), topical, oral, inhalation, rectal and transdermal.

The term “effective amount” includes an amount effective, at dosages andfor periods of time necessary, to achieve the desired result. Aneffective amount of compound may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound to elicit a desired response in the subject. Dosage regimensmay be adjusted to provide the optimum therapeutic response. Aneffective amount is also one in which any toxic or detrimental effects(e.g., side effects) of the elastase inhibitor compound are outweighedby the therapeutically beneficial effects.

The phrases “systemic administration,” “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound(s), drug or other material,such that it enters the patient's system and, thus, is subject tometabolism and other like processes.

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated.

A therapeutically effective amount of compound (I.e., an effectivedosage) may range from about 0.005 μg/kg to about 200 mg/kg, preferablyabout 0.1 mg/kg to about 200 mg/kg, more preferably about 10 mg/kg toabout 100 mg/kg of body weight. In other embodiments, thetherapeutically effect amount may range from about 1.0 pM to about 500nM. The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a compound can include a singletreatment or, preferably, can include a series of treatments. In oneexample, a subject is treated with a compound in the range of betweenabout 0.005 μg/kg to about 200 mg/kg of body weight, one time per weekfor between about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of a compound used for treatment may increase or decrease overthe course of a particular treatment.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate,”

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

The term “prodrug” includes compounds with moieties which can bemetabolized in vivo. Generally, the prodrugs are metabolized in vivo byesterases or by other mechanisms to active drugs. Examples of prodrugsand their uses are well known in the art (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugs can beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form or hydroxyl with a suitable esterifying agent. Hydroxyl groupscan be converted into esters via treatment with a carboxylic acid.Examples of prodrug moieties include substituted and unsubstituted,branch or unbranched lower alkyl ester moieties, (e.g., propionoic acidesters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,pivaloyloxymethyl ester), aryl esters (phenyl ester), acyl-lower alkylesters (e.g., benzyl ester), substituted (e.g., with methyl, halo, ormethoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferredprodrug moieties are propionoic acid esters and acyl esters. Prodrugswhich are converted to active forms through other mechanisms in vivo arealso included. In aspects, the compounds of the invention are prodrugsof any of the formulae herein.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

Furthermore the compounds of the invention include olefins having eithergeometry: “Z” refers to what is referred to as a “cis” (same side)conformation whereas “E” refers to what is referred to as a “trans”(opposite side) conformation. With respect to the nomenclature of achiral center, the terms “d” and “1” configuration are as defined by theIUPAC Recommendations. As to the use of the terms, diastereomer,racemate, epimer and enantiomer, these will be used in their normalcontext to describe the stereochemistry of preparations,

As used herein, the term “alkyl” refers to a straight-chained orbranched hydrocarbon group containing 1 to 12 carbon atoms. The term“lower alkyl” refers to a C1-C6 alkyl chain. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl.Alkyl groups may be optionally substituted with one or moresubstituents.

The term “alkenyl” refers to an unsaturated hydrocarbon chain that maybe a straight chain or branched chain, containing 2 to 12 carbon atomsand at least one carbon-carbon double bond. Alkenyl groups may beoptionally substituted with one or more substituents.

The term “alkynyl” refers to an unsaturated hydrocarbon chain that maybe a straight chain or branched chain, containing the 2 to 12 carbonatoms and at least one carbon-carbon triple bond. Alkynyl groups may beoptionally substituted with one or more substituents.

The sp² or sp carbons of an alkenyl group and an alkynyl group,respectively, may optionally be the point of attachment of the alkenylor alkynyl groups.

The term “alkoxy” refers to an —O-alkyl radical.

As used herein, the term “halogen”, “hal” or “halo” means —F, —Cl, —Bror —I.

The term “cycloalkyl” refers to a hydrocarbon 3-8 membered monocyclic or7-14 membered bicyclic ring system having at least one saturated ring orhaving at least one non-aromatic ring, wherein the non-aromatic ring mayhave some degree of unsaturation. Cycloalkyl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a cycloalkyl group may be substituted by asubstituent. Representative examples of cycloalkyl group includecyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and thelike.

The term “aryl” refers to a hydrocarbon monocyclic, bicyclic ortricyclic aromatic ring system. Aryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, 4, 5 or 6 atoms of each ring of an aryl group may be substituted by asubstituent. Examples of acyl groups include phenyl, naphthyl,anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, andthe remainder ring atoms being carbon (with appropriate hydrogen atomsunless otherwise indicated). Heteroaryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a heteroaryl group may be substituted by asubstituent. Examples of heteroaryl groups include pyridyl, furanyl,thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl,isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, indazolyl, and thelike.

The term “heterocycloalkyl” refers to a nonaromatic 3-8 memberedmonocyclic, 7-12 membered bicyclic, or 10-14 membered tricyclic ringsystem comprising 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, S, B, P or Si, wherein the nonaromatic ring system iscompletely saturated. Heterocycloalkyl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a heterocycloalkyl group may besubstituted by a substituent. Representative heterocycloalkyl groupsinclude piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl,thiomorpholinyl, 1,3-dioxolane, tetrahydrofuranyl, tetrahydrothienyl,thiirenyl, and the like.

The term “alkylamino” refers to an amino substituent which is furthersubstituted with one or two alkyl groups. The term “aminoalkyl” refersto an alkyl substituent which is further substituted with one or moreamino groups. The term “hydroxyalkyl” or “hydroxylalkyl” refers to analkyl substituent which is further substituted with one or more hydroxylgroups. The alkyl or aryl portion of alkylamino, aminoalkyl,mercaptoalkyl, hydroxyalkyl, mercaptoalkoxy, sulfonylalkyl,sulfonylaryl, alkylcarbonyl, and alkylcarbonylalkyl may be optionallysubstituted with one or more substituents.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

Alkylating agents are any reagent that is capable of effecting thealkylation of the functional group at issue (e.g., oxygen atom of analcohol, nitrogen atom of an amino group). Alkylating agents are knownin the art, including in the references cited herein, and include alkylhalides (e.g., methyl iodide, benzyl bromide or chloride), alkylsulfates (e.g., methyl sulfate), or other alkyl group-leaving groupcombinations known in the art. Leaving groups are any stable speciesthat can detach from a molecule during a reaction (e.g., eliminationreaction, substitution reaction) and are known in the art, including inthe references cited herein, and include halides (e.g., I—, Cl—, Br—,F—), hydroxy, alkoxy (e.g., —OMe, —O-t-Bu), acyloxy anions (e.g., —OAc,—OC(O)CF₃), sulfonates (e.g., mesyl, tosyl), acetamides (e.g.,—NHC(O)Me), carbamates (e.g., N(Me)C(O)Ot-Bu), phosphonates (e.g.,—OP(O)(OEt)₂), water or alcohols (protic conditions), and the like.

In certain embodiments, substituents on any group (such as, for example,alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkyl, heterocycloalkyl) can be at any atom of that group, whereinany group that can be substituted (such as, for example, alkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,heterocycloalkyl) can be optionally substituted with one or moresubstituents (which may be the same or different), each replacing ahydrogen atom. Examples of suitable substituents include, but are notlimited to alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaralkyl, aryl, heteroaryl, halogen, haloalkyl, cyano,nitro, alkoxy, aryloxy, hydroxyl, hydroxylalkyl, oxo (i.e., carbonyl),carboxyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl,alkylcarbonyloxy, aryloxycarbonyl, heteroaryloxy, heteroaryloxycarbonyl,thio, mercapto, mercaptoalkyl, arylsulfonyl, amino, aminoalkyl,dialkylamino, alkylcarbonylamino, alkylaminocarbonyl,alkoxycarbonylamino, alkylamino, arylamino, diarylamino, alkylcarbonyl,or arylamino-substituted aryl; arylalkylamino, aralkylaminocarbonyl,amide, alkylaminosulfonyl, arylaminosulfonyl, dialkylaminosulfonyl,alkylsulfonylamino, arylsulfonylamino, imino, carbamido, carbamyl,thioureido, thiocyanato, sulfoamido, sulfonylalkyl, sulfonylaryl, ormercaptoalkoxy.

Compounds of the Invention and Structure Elucidation

A sample of Symploca sp. was collected from Key Largo, Florida Keys andextracted with organic solvents. The resulting cytotoxic crude extractwas subjected to bioassay-guided fractionation by solvent partition,silica gel chromatography and reversed-phase HPLC to yield largazole (1)as a colorless, amorphous solid {[α]²⁰ _(D)+22 (c 0.1, MeOH)},

¹H and ¹³C NMR data coupled with a [M+H]⁺ peak at m/z 623.2397 in theHR-ESI/APCI-MS of 1 suggested a molecular formula of C₂₉H₄₂N₄O₅S₃ (calcdfor C₂₉H₄₃N₄O₅S₃, 623.2396). The ¹H NMR spectrum exhibited two signalscharacteristic for secondary amides (δ_(2-NH) 7.15, δ_(14-NH) 6.45).Further two-dimensional NMR analysis in CDCl₃ using COSY, HSQC and HMBCdata indicated that these exchangeable protons belong to valine andmodified glycine residues, respectively (Table 1 and SupportingInformation). The putative glycine carbonyl (δ_(C-13) 167.9) was part ofa 2,4-disubstituted thiazole unit as evidenced by HMBCs from the onlyaromatic methine (δ_(H-12) 7.76, δ_(C-12) 124.2) to C-13 and to anotherquaternary sp² carbon, C-11 (δ_(C) 147.4). Furthermore, HMBCs from amethyl singlet (δ_(H-9) 1.87) to carbonyl C-6 (δ_(C) 173.5), quaternarycarbon C-7 (δ_(C) 84.4) and methylene carbon C-8 (δ_(C) 43.3), combinedwith an HMBC from H-8a (δ_(H) 4.04) to C-10 (δ_(C) 164.6) suggested thepresence of a 2-substituted thiazoline-4-methyl-4-carboxylic acid unit(C-6 to C-10). The only other HMBC to C-10 was from the thiazole protonH-12, indicating that C-10 bore the thiazole substituent. The methylthiazoline carboxylate and the amino terminus of the valine residue wereunambiguously connected via an amide linkage based on HMBC data (Table1). The remaining signals in the ¹H NMR spectrum belonged to two spinsystems, as concluded from COSY analysis (Supporting Information). Oneof the units was a 7-substituted 3-hydroxy-hept-4-enoic acid moiety(C-15 to C-21) with E-geometry of the double bond based on a largecoupling constant for ³J_(H-18,H-19) of 15.6 Hz, consistent with NOESYcross peaks between H-18 and H₂-20. This unit was attached to the aminoterminus of the glycine-derived unit as shown by HMBCs from 14-NH andH-14a/b to C-15 as well as ROESY cross peaks between 14-NH and H-16a andH-16b. The last unit was an n-octanoyl group (C-22 to C-29) which wasconnected with C-21 based on HMBC from H₂-21 to C-22. The low-fieldchemical shift for C-22 (δ_(C) 199.4) coupled with the fact that onesulfur atom yet remained to be assigned was strong evidence for athioester functionality. Finally, to account for the molecular formularequirements and for the low-field chemical shift of H-17 (δ_(H) 5.66)suggestive of an acyloxy substituent, C-17 had to be ester-linked to thecarboxyl terminus of valine. This was further supported by a weak NOEbetween H-17 and H₃-5 (δ_(H) 0.50), leading to the cyclic planarstructure shown for 1.

To assign the absolute configuration of the three chiral centers, ourstrategy was to generate optically active fragments, for whichenantiomeric standards are readily available (Scheme 1). Specifically,ozonolysis followed by oxidative work-up and acid hydrolysis generated2-methylcysteic acid, valine and malic acid. The product mixture wassubjected to chiral HPLC analysis, comparing retention times with thoseof authentic standards. This analysis identified L-valine,(R)-2-methylcysteic acid and L-malic acid, establishing the absoluteconfiguration of 1 as 2S,7R,17S.

TABLE 1 NMR Spectral Data for Largazole (1) in CDCl₃ (600 MHz) C/H no.δ_(H) (J in Hz) δ_(C), mult. HMBC^(a,b)  1 168.9, qC  2 4.61, dd (9.2,3.3) 57.7, CH 1, 3, 4, 5, 6  3 2.10, m 34.2, CH 1,^(c) 2^(c)  4 0.68, d(7.2) 18.9, CH₃ 2, 3, 5  5 0.50, d (7.2) 16.6, CH₃ 2, 3, 4  2-NH 7.15, d(9.2) 1, 6^(c)  6 173.5, qC  7 84.4, qC  8a 4.04, d (−11.4) 43.3, CH₂ 6,7, 10  8b 3.27, d (−11.4) 6, 7, 9  9 1.87, br s 24.2, CH₃ 6, 7, 8 10164.6, qC 11 147.4, qC 12 7.76, s 124.2, CH 10,^(c) 11, 13 13 167.9, qC14a 5.29, dd (−17.4, 9.6) 41.1, CH 13, 15 14b 4.27, dd (−17.4, 2.5) 13,15 14-NH 6.45, dd (9.6, 2.5) 15^(c) 15 169.4, qC 16a 2.86, dd (−16.5,10.5) 40.5, CH₂ 15, 17, 18 16b 2.68, dd (−16.5, 1.8) 15 17 5.66, ddd(10.5, 7.2, 1.8) 72.0, CH 18 5.51, dd (15.6, 7.2) 128.4, CH 17, 20 195.82, dt (15.6, 7.2) 132.7, CH 17, 20 20 2.31, br q (7.2) (2H) 32.3, CH₂18, 19, 21 21 2.90, t (7.2) (2H) 27.9, CH₂ 19, 20, 22 22 199.4, qC 232.52, t (7.5) (2H) 44.1, CH₂ 22, 24, 25 24 1.64, m (2H) 25.6, CH₂ 22,23, 25/26 25 1.29, m (2H) 28.9, CH₂ 26 26 1.25, m (2H) 28.9, CH₂ 25, 2727 1.26, m (2H) 31.6, CH₂ 28 1.28, m (2H) 22.6, CH₂ 29 0.87, br t (6.9)14.0, CH₃ 27, 28 ^(a)Protons showing HMBC correlations to the indicatedcarbon. ^(b)Optimized for ″J = 7 Hz if not indicated otherwise.^(c)Optimized for ″J = 3.5 Hz.

Largazole (1) possesses a dense combination of unusual structuralfeatures, including a substituted 4-methylthiazoline linearly fused to athiazole, previously only found in didehydromirabazole,⁷ a member of thegroup of terrestrial cyanobaeterial cytotoxins from Scytonema mirabilewith solid tumor selectivity.⁸ Another remarkable structural element isthe thioester moiety; thioester-containing secondary metabolites havebeen reported previously from sponges,⁹ eukaryotic algae¹⁰ andbacteria,¹¹ but not from cyanobacteria. The3-hydroxy-7-thio-hept-4-enoic acid unit in 1 is unprecedented in naturalproducts. Most significantly, the potent biological activity andselectivity for cancer cells warrants further investigation as to themode of action, cancer chemotherapeutic potential and biosynthesis oflargazole (1).

Compounds of the invention can be made by means known in the art oforganic synthesis. Methods for optimizing reaction conditions, ifnecessary minimizing competing by-products, are known in the art.Reaction optimization and scale-up may advantageously utilize high-speedparallel synthesis equipment and computer-controlled microreactors (e.g.Design And Optimization in Organic Synthesis, 2^(nd) Edition, Carlson R,Ed, 2005; Elsevier Science Ltd.; Jähnisch, K et al, Angew. Chem. Int.Ed. Engl. 2004 43: 406; and references therein). Additional reactionschemes and protocols may be determined by the skilled artesian by useof commercially available structure-searchable database software, forinstance, SciFinder® (CAS division of the American Chemical Society) andCrossFire Beilstein® (Elsevier MDL), or by appropriate keyword searchingusing an internet search engine such as Google® or keyword databasessuch as the US Patent and Trademark Office text database.

The compounds herein may also contain linkages (e.g., carbon-carbonbonds) wherein bond rotation is restricted about that particularlinkage, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans and E/Z isomers are expresslyincluded in the present invention. The compounds herein may also berepresented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein, even though only a single tautomeric form may berepresented. All such isomeric forms of such compounds herein areexpressly included in the present invention. All crystal forms andpolymorphs of the compounds described herein are expressly included inthe present invention. Also embodied are extracts and fractionscomprising compounds of the invention. The term isomers is intended toinclude diastereoisomers, enantiomers, regioisomers, structural isomers,rotational isomers, tautomers, and the like. For compounds which containone or more stereogenic centers, e.g., chiral compounds, the methods ofthe invention may be carried out with an enantiomerically enrichedcompound, a racemate, or a mixture of diastereomers.

Preferred enantiomerically enriched compounds have an enantiomericexcess of 50% or more, more preferably the compound has an enantiomericexcess of 60%, 70%, 80%, 90%, 95%, 98%, or 99% or more. In preferredembodiments, only one enantiomer or diastereomer of a chiral compound ofthe invention is administered to cells or a subject.

Methods of Treatment

The invention is directed towards macrocyclic compounds, and methods oftreating disease and disorders using the compounds or compositionsthereof delineated herein.

In other aspects, the invention provides a method of treating a subjectsuffering from or susceptible to HDAC related disorder or disease,wherein the subject has been identified as in need of treatment for aHDAC related disorder or disease, comprising administering to saidsubject in need thereof, an effective amount of a compound orpharmaceutical composition of formula I, such that said subject istreated for said disorder. Identifying a subject in need of suchtreatment can be in the judgment of a subject or a health careprofessional and can be subjective (e.g. opinion) or objective (e.g.measurable by a test or diagnostic method).

In one aspect, the invention provides a method of modulating theproliferation activity of a cell in a subject, comprising contacting thesubject with a compound of formula I, in an amount and under conditionssufficient to modulate cell proliferation activity.

In one embodiment, the modulation is inhibition.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a cell proliferation related disorderor disease, comprising administering to the subject an effective amountof a compound or pharmaceutical composition of formula I.

In other aspects, the invention provides a method of treating a subjectsuffering from or susceptible to a cell proliferation related disorderor disease, wherein the subject has been identified as in need oftreatment for a cell proliferation related disorder or disease,comprising administering to said subject in need thereof, an effectiveamount of a compound or pharmaceutical composition of formula I, suchthat said subject is treated for said disorder.

In certain embodiments, the invention provides a method as describedabove, wherein the compound of formula I is largazole.

In certain embodiments, the invention provides a method of treating adisorder, wherein the disorder is cancer (e.g., breast, colon) or solidtumor.

In certain embodiments, the subject is a mammal, preferably a primate orhuman.

In another embodiment, the invention provides a method as describedabove, wherein the effective amount of the compound of formula I rangesfrom about 0.005 μg/kg to about 200 mg/kg. In certain embodiments, theeffective amount of the compound of formula I ranges from about 0.1mg/kg to about 200 mg/kg. In a further embodiment, the effective amountof compound of formula I ranges from about 10 mg/kg to 100 mg/kg.

In other embodiments, the invention provides a method as described abovewherein the effective amount of the compound of formula I ranges fromabout 1.0 pM to about 500 nM. In certain embodiments, the effectiveamount ranges from about 10.0 pM to about 1000 pM. In anotherembodiment, the effective amount ranges from about 1.0 nM to about 10nM.

In another embodiment, the invention provides a method as describedabove, wherein the compound of formula I is administered intravenously,intramuscularly, subcutaneously, intracerebroventricularly, orally ortopically.

In another embodiment, the invention provides a method as describedherein wherein the compound of formula I demonstrates selectivity (e.g.,at least 2-fold, at least 5-fold, at least 10-fold, at least X-foldwhere X is any number between 1 and 20 inclusive) in cell growthactivity (e.g., in transformed/nontransformed, MDA-MB-231/NMuMG,U2OS/NIH3T3 cells). In another aspect, the compound of formula Idemonstrates selectivity in modulating cell growth activity (e.g., atleast 2-fold, at least 5-fold, at least 10-fold, at least X-fold where Xis any number between 1 and 20 inclusive) relative to another standardanticancer therapy (e.g., paclitaxel, actinomycin D, doxorubicin).

In other embodiments, the invention provides a method as describedabove, wherein the compound of formula I is administered alone or incombination with one or more other therapeutics. In a furtherembodiment, the additional therapeutic agent is an anti-cancer agent,chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, oran anti-proliferation agent. Examples of such chemotherapeutic agentsinclude but are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds.,Rahay, N.J., 1987).

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a cell proliferation disorder ordisease, or to affect cell differentiation, dedifferentiation ortransdifferentiation. Another object of the present invention is the useof a compound as described herein (e.g., of any formulae herein) for usein the treatment of a cell proliferation disorder or disease, or affectcell differentiation, dedifferentiation or transdifferentiation.

Pharmaceutical Compositions

In one aspect, the invention provides a pharmaceutical compositioncomprising the compound of formula I and a pharmaceutically acceptablecarrier.

In one embodiment, the invention provides a pharmaceutical compositionwherein the compound of formula I is largazole, and a pharmaceuticallyacceptable carrier.

In another embodiment, the invention provides a pharmaceuticalcomposition further comprising an additional therapeutic agent. In afurther embodiment, the additional therapeutic agent is an anti-canceragent, chemotherapeutic agent, an anti-angiogenesis agent, cytotoxicagent, or an anti-proliferation agent.

In one aspect, the invention provides a kit comprising an effectiveamount of a compound of formula I, in unit dosage form, together withinstructions for administering the compound to a subject suffering fromor susceptible to a HDAC mediated disease or disorder, including memoryloss, inducing neurogenesis, enhancing memory retention, enhancingmemory formation, increasing synaptic potential or transmission, orincreasing long term potentiation (LTP), etc.

In one aspect, the invention provides a kit comprising an effectiveamount of a compound of formula I, in unit dosage form, together withinstructions for administering the compound to a subject suffering fromor susceptible to a cell proliferation disease or disorder, includingcancer, solid tumor, angiogenesis, etc.

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, e.g., Berge et al.,Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Other pharmaceutically acceptable carriersknown to those of skill in the art are suitable for the presentinvention.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

The invention also provides a pharmaceutical composition, comprising aneffective amount a compound described herein and a pharmaceuticallyacceptable carrier. In an embodiment, compound is administered to thesubject using a pharmaceutically-acceptable formulation, e.g., apharmaceutically-acceptable formulation that provides sustained deliveryof the compound to a subject for at least 12 hours, 24 hours, 36 hours,48 hours, one week, two weeks, three weeks, or four weeks after thepharmaceutically-acceptable formulation is administered to the subject.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of this invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic(or unacceptably toxic) to the patient.

In use, at least one compound according to the present invention isadministered in a pharmaceutically effective amount to a subject in needthereof in a pharmaceutical carrier by intravenous, intramuscular,subcutaneous, or intracerebro ventricular injection or by oraladministration or topical application. In accordance with the presentinvention, a compound of the invention may be administered alone or inconjunction with a second, different therapeutic. By “in conjunctionwith” is meant together, substantially simultaneously or sequentially.In one embodiment, a compound of the invention is administered acutely.The compound of the invention may therefore be administered for a shortcourse of treatment, such as for about 1 day to about 1 week. In anotherembodiment, the compound of the invention may be administered over alonger period of time to ameliorate chronic disorders, such as, forexample, for about one week to several months depending upon thecondition to be treated.

By “pharmaceutically effective amount” as used herein is meant an amountof a compound of the invention, high enough to significantly positivelymodify the condition to be treated but low enough to avoid serious sideeffects (at a reasonable benefit/risk ratio), within the scope of soundmedical judgment. A pharmaceutically effective amount of a compound ofthe invention will vary with the particular goal to be achieved, the ageand physical condition of the patient being treated, the severity of theunderlying disease, the duration of treatment, the nature of concurrenttherapy and the specific organozinc compound employed. For example, atherapeutically effective amount of a compound of the inventionadministered to a child or a neonate will be reduced proportionately inaccordance with sound medical judgment. The effective amount of acompound of the invention will thus be the minimum amount which willprovide the desired effect.

A decided practical advantage of the present invention is that thecompound may be administered in a convenient manner such as byintravenous, intramuscular, subcutaneous, oral orintra-cerebroventricular injection routes or by topical application,such as in creams or gels. Depending on the route of administration, theactive ingredients which comprise a compound of the invention may berequired to be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound. In order to administer a compound of theinvention by other than parenteral administration, the compound can becoated by, or administered with, a material to prevent inactivation.

The compound may be administered parenterally or intraperitoneally.Dispersions can also be prepared, for example, in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage. The carrier can be asolvent or dispersion medium containing, for example, water, DMSO,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion. In many cases it willbe preferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized compounds into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and the freeze-drying techniquewhich yields a powder of the active ingredient plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

For oral therapeutic administration, the compound may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Compositions or preparations according to the presentinvention are prepared so that an oral dosage unit form containscompound concentration sufficient to treat a disorder in a subject.

Some examples of substances which can serve as pharmaceutical carriersare sugars, such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethycellulose, ethylcellulose and cellulose acetates; powderedtragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil,sesame oil, olive oil, corn oil and oil of theobroma; polyols such aspropylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol;agar; alginic acids; pyrogen-free water; isotonic saline; and phosphatebuffer solution; skim milk powder; as well as other non-toxic compatiblesubstances used in pharmaceutical formulations such as Vitamin C,estrogen and echinacea, for example. Wetting agents and lubricants suchas sodium lauryl sulfate, as well as coloring agents, flavoring agents,lubricants, excipients, tableting agents, stabilizers, anti-oxidants andpreservatives, can also be present.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

EXAMPLES

The present invention will now be demonstrated using specific examplesthat are not to be construed as limiting.

General Experimental Procedures

¹H and ¹³C NMR data were acquired on a Bruker Avance 600 MHzspectrometer with a 5-mm probe operating at 600 and 150 MHz,respectively. 2D NMR data were recorded on a Bruker Avance II 600 MHzequipped with a 1-mm triple resonance high-temperature superconductingcryogenic probe using residual solvent signals (δ_(H) 7.26 ppm, δ_(C)77.0 ppm) as internal standards. The HSQC experiments were optimized for¹J_(CH)=145 Hz, and the HMBC experiments for ¹J_(CH)=7 or 3.5 Hz. LC-MSdata were obtained using an Agilent 1100 equipped with a ThermoFinniganLCQ by ESI (positive mode). HRMS data were obtained using an AgilentLC-TOF mass spectrometer equipped with an ESI/APCI multimode ion sourcedetector. Enantiomeric standards of 2-methylcysteic acid were obtainedby oxidation of (R)- and (S)-2-methylcysteines (see below) which wereprovided by ResCom (DSM Pharma Chemicals). Valine, glycine and malicacid standards were obtained from Sigma. Paclitaxel, actinomycin D anddoxorubicin were obtained from EMD Chemicals, Inc.

Example 1 Extraction and Isolation

A sample of Symploca sp. was collected from Pillars, Key Largo (FloridaKeys, USA) in August 2003. The specimens had upright, golden-brown,feather-like filaments consistent with this genus. Filaments measured5-6 μm in width including a fine sheath and 8-9 μm in length. Symplocasp. was freeze-dried and extracted with MeOH-EOAc (1:1). The resultinglipophilic extract (0.29 g) was partitioned between hexanes and 20%aqueous MeOH. The aqueous MeOH layer was concentrated and fractionatedby Si gel chromatography using CH₂Cl₂ containing increasing amounts ofi-PrOH followed by MeOH. The fraction that eluted with 5% i-PrOH wasthen subjected to reversed-phase HPLC(YMC-pack ODS-AQ, 250×10 mm, 2.0mL/min; detection at 220 and 254 nm) using a MeOH-H₂O linear gradient(40-100% for 75 min and then 100% MeOH for 10 min). Compound 1 eluted att_(R) 61.5 min (1.2 mg).

Largazole (1): colorless, amorphous solid; [α]²⁰ _(D)+22 (c 0.1, MeOH);UV (MeOH) (log ε) 210 (4.07), 260 (sh) (3.61); IR (film) v_(max) 2924,2853, 1725, 1694, 1611, 1659, 1641, 1630, 1596, 1512, 1249, 1117, 1067,1034, 894 cm⁻¹; ¹H NMR, ¹³C NMR, and HMBC data, see Table 1;HR-ESI/APCI-MS m/z [M+H]⁺623.2397 (calcd for C₂₉H₄₃N₄O₅S₃ 623.2396).

LC-MS^(n) Analysis. A sample of compound 1 was analyzed by LC-MS[column: Waters Corp., Atlantis dC18 3 μm, 2.1×150 mm; mobile phase:0.5% HCOOH in MeOH (A) in 0.5% HCOOH in H₂O (B); flow rate: 0.15 mL/min]using a linear gradient (5-95% for 65 min). (+) ESI-MS (m/z 200-1600) ofthe most intense ion of the MS range (t_(R) 51.1 min, m/z 623) as wellas MS/MS and dependent MS/MS/MS of the m/z 623 [M+H]⁺ ion was carriedout (See, FIG. 1).

Example 2 Synthesis of (R)- and (S)-2-methylcysteic acid

A sample of (R)-2-methylcysteine (5.0 mg) was treated with 2 mL of amixture of H₂O₂—HCO₂H (1:9) at 0° C. for 2 h. The product mixture wasconcentrated to dryness by evaporation to give (R)-2-methylcysteic acid.The residue was then reconstituted in 250 μL of H₂O for amino acidanalysis by chiral HPLC. Similarly, (S)-2-methylcysteine was reacted toyield (S)-2-methylcysteic acid.

Example 3 Determination of Absolute Configuration

A sample of compound 1 (˜100 μg) was dissolved in 4 mL of CH₂Cl₂ andsubjected to ozonolysis at room temperature for 30 min. The solvent wasevaporated and the residue was treated with 0.6 mL of H₂O₂—HCO₂H (1:2)at 70° C. for 20 min. The solvent was evaporated and the resultingoxidation product was hydrolyzed with 0.5 mL of 6 N HCl at 110° C. for24 h. The hydrolyzed product was dried and analyzed by chiral HPLC(column, Phenomenex Chirex phase 3126 N,S-dioctyl-(D)-pencillamine,4.60×250 mm, 5 μm; solvent 1, 2 mM CuSO₄ in 95:5 H₂O/MeCN, pH 4.50;solvent 2, 0.5 mM Cu(OAc)₂/0.1 M NH₄OAc in 85:15 H₂O/MeCN, pH 4.6; flowrate 1.0 mL/min; detection at 254 nm). The absolute configuration of theamino acids in the hydrolyzate was determined by direct comparison withthe retention times of authentic standards. The retention times (t_(R),min) for solvent 1 were as follows: Gly (5.3), L-Val (12.6), D-Val(16.4), (S)-2-Me-cysteic acid (20.0), and (R)-2-Me-cysteic acid (23.9).The retention times (t_(R), min) of the hydrolyzate components were 5.3,12.6, 23.9, indicating the presence of Gly, L-Val and (R)-2-Me-cysteicacid in the product mixture. Solvent 2 was used to detect malic acid.Standard L-malic acid eluted at t_(R) 7.6 min and 1)-malic acid at t_(R)20.4 min. Malic acid in the hydrolyzate eluted after 7.6 min, indicatingthe presence of the L isomer. Gly, L-Val, and (R)-2-Me-cysteic acideluted after 4.0, 5.8 and 6.5 min, respectively.

Example 4 Cell Culture

Cell culture medium was purchased from Invitrogen and fetal bovine serum(FBS) from Hyclone. Cells were propagated and maintained in DMEM medium(high glucose) supplemented with 10% FBS at 37° C. humidified air and 5%CO₂.

Example 5 Cell Viability Assays

Cells suspended in DMEM containing 10% FBS were plated in 96-well plates(MDA-MB-231: 12,000 cells; NMuMG: 5,000 cells; U2OS: 5,000 cells; HT29:10,000 cells; IMR-32: 30,000 cells; NIH3T3: 5,000 cells) incubated (37°C., 5% CO₂) and 24 h later treated with various concentrations ofcompound 1 or solvent control (1% EtOH). After another 48 h ofincubation, cell viability was measured using MTT according tomanufacturer's instructions (Promega).

Example 6 Anticancer Therapeutics Activity

MDA-MB-231 and NMuMG cells were also treated with paclitaxel (in DMSO),actinomycin D (in DMSO) and doxorubicin (in H₂O) and correspondingsolvent control (1%) in the same manner. GI₅₀ and LC₅₀ values werecalculated as previously described (K. D. Paull, E. Hamel, L. Malspeis,In Cancer Chemotherapeutic Agents, W. E. Foye, Ed., American ChemicalSociety, Washington, D.C., 1995, pp. 10-11).

GI₅₀: concentration where

${{100 \times \frac{( {T - T_{0}} )}{( {C - T_{0}} )}} = 50};$

LC₅₀: concentration where

${100 \times \frac{( {T - T_{0}} )}{T_{0}}} = {- 50.}$

[T=absorbance in treated wells (48 h); T₀=absorbance at time zero;C=absorbance in control wells (48 h)]

Example 7 Largazole Activity

Largazole (1) potently inhibited the growth of highly invasivetransformed human mammary epithelial cells (MDA-MB-231) in adose-dependent manner (GI₅₀ 8 nM) and induced cytotoxicity at higherconcentrations (LC₅₀ 117 nM) based on MTT assay. In contrast,nontransformed murine mammary epithelial cells (NMuMG) weresignificantly less susceptible to compound 1 (GI₅₀ 122 nM, LC₅₀ 272 nM).Similarly, while fibroblastic osteosarcoma U2OS cells were highlysusceptible to largazole (1) with a GI₅₀ of 55 nM and LC₅₀ of 94 nM, theviability of nontransformed fibroblasts NIH3T3 upon treatment with 1 wassignificantly less compromised (GI₅₀ 480 nM) with no apparent toxicity.The 8- to 15-fold differential growth-inhibitory activity betweentransformed and nontransformed fibroblasts or epithelial cells,respectively, and selectivity for killing transformed fibroblasts overnontransformed fibroblasts suggests that cancer cells are preferentiallytargeted by 1. The growth of cancer cell lines derived from colon (HT29)and neuroblastoma (IMR-32) was also strongly inhibited by 1 (GI₅₀ valuesof 12 nM and 16 nM, respectively), accompanied by cytotoxicity (LC₅₀ 22nM for both cell lines).

Largazole (1) also demonstrates remarkable selectivity that is notobserved with other validated antitumor natural products tested inparallel. See, e.g., Table 2 re MDA-MB-231/NmuMG cells and U2OS/NIH3T3cells.

TABLE 2 Growth-inhibitory activity (GI₅₀) of natural product drugsCompound MDA-MB-231 NMuMG U2OS NIH3T3 Largazole (1) 7.7 nM 122 nM  55 nM480 nM  Paclitaxel 7.0 nM 5.9 nM 12 nM 6.4 nM Actinomycin D 0.5 nM 0.3nM 0.8 nM  0.4 nM Doxorubictn 310 nM   63 nM 220 nM   47 nM

Example 8 HDAC Inhibition

To test this hypothesis we determined the cellular HDAC activity upontreatment with largazole in HCT-116 cells found to possess highintrinsic HDAC activity. We co-incubated a cell-permeable fluorogenicartificial HDAC substrate (fluor de Lys™, BIOMOL) and largazole (1) anddetermined that largazole treatment for 8 h resulted in a decrease ofHDAC activity in a dose-response manner (FIG. 2 a) and, importantly, theIC₅₀ for HDAC inhibition closely corresponded with the GI₅₀ of largazolein this cell line (FIG. 2 a, Table 3). This correlation suggested thatHDAC is the relevant target responsible for largazole'santiproliferative effect. Confirmatory, immunoblot analysis of anendogenous HDAC substrate, acetylated histone H3, revealed the samedose-response relationship (FIG. 2 b).

Largazole (1) inhibited HDAC activity from a HeLa cell nuclear proteinextract rich in class I HDACs 1, 2, and 3 (BIOMOL); however, it ispossible that the thioester is cleaved under assay conditions. Toinvestigate that thiol 9 is a reactive species, we liberated 9 from theacetyl analog 8 of largazole (1) and measured enzymatic activitydirectly; thiol 9 inhibited the HDACs in the nuclear extract of HeLacells with a similar IC₅₀ value (Table 3). Largazole (1) and thiol 9exhibited similar cellular activity against HDACs derived from nuclearHeLa extracts as well as antiproliferative activity.

TABLE 3 IC₅₀ and GI₅₀ Values for HDAC and Growth Inhibition (nM) HCT-116HCT-116 HeLa nuclear growth HDAC extract inhibition cellular assay HDACs1  44 ± 10 51 ± 3 37 ± 11 8 33 ± 2  50 ± 18 52 ± 27 9 38 ± 5 209 ± 15 42± 29

In vitro cellular HDAC activity assay. HCT-116 cells are seeded in 100μL of medium per well at a density of 3.5×10³ cells/well and grown for24 h in a sterile 96-well solid bottom plate. The assay is carried outaccording to the manufacturer's instructions (BIOMOL). Briefly, after 24h the medium is replaced with 50 μL/well of medium containing 200 μMfluor de Lys™ substrate and 1 μM of trichostatin A (positive control)and test compounds (largazole, acetyl derivative) ranging from 1 μM to3.2 nM (1 g/2-fold dilutions) and thiol compound ranging from 10 μM to300 pM. Plates are incubated at 37° C. for 8 h. After the treatmenttime, 50 μL per well of 1× fluor de Lys™ Developer containing TSA at 2μM is added. After developer addition, plates are incubated for 15 minat 37° C. and fluorescence is read (Ex 360 nm, Em 460 nm).

Immunoblot analysis. HCT-116 cells (650,000 cells/dish) are seeded in10-cm dishes and 24 h later treated with various concentrations oflargazole, trichostatin or solvent controls (EtOH for largazole and DMSOfor trichostatin). Following incubation for 8 h, whole-cell lysates areprepared using PhosphoSafe lysis buffer (Novagen), proteins extracted,and protein concentration measured using the BCA method (Pierce). Celllysates containing equal amounts of protein are separated by SDS-PAGE,transferred to PVDF membranes, probed with antibodies and detected withthe Supersignal Femto Western blotting kit (Pierce). Anti-acetyl-histoneH3 (Lys9/18) and anti-histone H3 primary antibodies are obtained fromMillipore and horseradish peroxidase conjugated anti-rabbit secondaryantibody is purchased from Cell Signaling.

Cell-free enzymatic assay. Assay buffer (25 ul in blank and 10 ul incontrol), 1 μM trichostatin and test inhibitors (ranging from 10 μM to300 pM) are added to test sample wells of the microtiter plate.HDAC-enriched nuclear protein extract from HeLa cells (BIOMOL) (5 μg in15 uL) is added to all wells except in the no-enzyme control. The assayplate is equilibrated at 37° C., and then 25 μl of substrate fluor deLys™ substrate is added to a final concentration of 116 μM. HDACreaction is allowed to proceed for 15 min and then stopped by additionof 50 μl per well of 1× fluor de Lys™ Developer containing TSA at 2 μM.After developer addition, plates are incubated for 15 min at 37° C. andfluorescence is read (Ex 360 nm, Em 460 nm).

HDAC assay using purified enzymes. Similarly as above using HeLa nuclearextract as a source of HDAC activity, the assays can be executed usingdifferent pure HDACs of class I and class II to assess specificity.

Cell differentiation/dedifferentiation/transdifferetiation assays. Thecompounds herein are assessed for their ability to induce celldifferentiation/dedifferentiation/transdifferentiation by subjectingthem to assay conditions essentially as described in protocols known inthe art, including for example, as those described in Wu et al. J. Am.Chem. Soc. 2002, 124, 14520-14521; Ding et al. Proc. Natl. Acad. Sci.USA, 2003, 100, 7632-7637; and Chen et al. J. Am. Chem. Soc. 2004, 126,410-411.

REFERENCES

-   (1) (a) Koehn, F. E.; Carter, G. T. Nat. Rev. Drug Discov. 2005, 4,    206-220. (b) Paterson, I.; Anderson, E. A. Science 2005, 310,    451-453.-   (2) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666-673.-   (3) Gerwick, W. H.; Tan, L. T.; Sitachitta, N. Alkaloids Chem. Biol.    2001, 57, 75-184.-   (4) Luesch, H.; Moore, R. E.; Paul, V. J.; Mooberry, S. L.;    Corbett, T. H. J. Nat. Prod. 2001, 64, 907-910.-   (5) (a) Gerwick, W. H.; Protean, P. J.; Nagle, D. G.; Hamel, E.;    Blokhin, A.; Slate, D. L. J. Org. Chem. 1994, 59, 1243-1245. (b)    Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.;    Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J. R.; Gerwick, W. H.;    Day, B. W.; Hamel, E. Mol. Pharmacol. 1998, 53, 62-76.-   (6) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.;    Corbett, T. H. J. Am. Chem. Soc. 2001, 123, 5418-5423. (b) Luesch,    H.; Chanda, S. K.; Raya, M. R.; DeJesus, P. D.; Orth, A. P.;    Walker, J. R.; Izpistúa Belmonte, J. C.; Schultz, P. G. Nat. Chem.    Biol. 2006, 2, 158-167.-   (7) (a) Carmeli, S.; Moore, R. E.; Patterson, G. M. L. Tetrahedron    Lett 1991, 32, 2593-2596. (b) Pattenden, G.; Thom, S. M. J. Chem.    Soc. Perkin Trans. 11993, 1629-1636. (c) Boyce, R. J.; Pattenden, G.    Tetrahedron 1995, 51, 7313-7320.-   (8) Carmeli, S.; Moore, R. E.; Patterson, G. M. L. J. Am. Chem. Soc.    1990, 112, 8195-8197.-   (9) Horton, P.; Inman, W. D.; Crews, P. J. Nat. Prod. 1990, 53,    143-151.-   (10) (a) Roller, P.; Au, K.; Moore, R. E. Chem. Commun. 1971,    503-504. (b) Sata, N.; Abinsay, H.; Yoshida, W. Y.; Horgen, F. D.;    Sitachitta, N.; Kelly, M.; Scheuer, P. J. J. Nat. Prod. 2005, 68,    1400-1403.-   (11) (a) Perez Baz, J.; Cañedo, L. M.; Fernández, Puentes, J. L.;    Silva Elipe, M. V. J. Antibiot. (Tokyo) 1997, 50, 738-741. (b)    Boger, D. G.; Ichikawa, S. J. Am. Chem. Soc. 2000, 122, 2956-2957.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

1-11. (canceled)
 12. A kit comprising an effective amount of a compoundof formula (I), in unit dosage form, together with instructions foradministering the compound to a subject suffering from or susceptible toa cell proliferation disorder:

wherein: each R is independently H or optionally substituted alkyl; eachR¹ is independently H, or optionally substituted alkyl; each R² isindependently H, optionally substituted alkyl, or C(O)R; each R³ isindependently H, optionally substituted alkyl, C(O)OR, or C(O)NRR; eachR⁴ is independently H, optionally substituted alkyl, C(O)OR, or C(O)NRR;and pharmaceutically acceptable salts, solvate, or hydrate thereof. 13.A method of modulating the activity of cell proliferation in a subject,comprising contacting the subject with a compound of formula I, in anamount and under conditions sufficient to modulate cell proliferation:

wherein: each R is independently H or optionally substituted alkyl; eachR¹ is independently H, or optionally substituted alkyl; each R² isindependently H, optionally substituted alkyl, or C(O)R; each R³ isindependently H, optionally substituted alkyl, C(O)OR, or C(O)NRR; eachR⁴ is independently H, optionally substituted alkyl, C(O)OR, or C(O)NRR;and pharmaceutically acceptable salts, solvate, or hydrate thereof. 14.The method of claim 13, wherein the cell is a cancer cell.
 15. Themethod of claim 13, wherein the cell is a tumor cell.
 16. The method ofclaim 13, wherein the modulation is inhibition.
 17. A method of treatinga subject suffering from or susceptible to a cell proliferation relateddisorder or disease, wherein the subject has been identified as in needof treatment for a cell proliferation related disorder or disease,comprising administering to said subject in need thereof, an effectiveamount of a compound or pharmaceutical composition of formula I, suchthat said subject is treated for said disorder:

wherein: each R is independently H or optionally substituted alkyl: eachR¹ is independently H, or optionally substituted alkyl; each R² isindependently H, optionally substituted alkyl, or C(O)R; each R³ isindependently H, optionally substituted alkyl, C(O)OR, or C(O)NRR; eachR⁴ is independently H, optionally substituted alkyl, C(O)OR, or C(O)NRR;and pharmaceutically acceptable salts, solvate, or hydrate thereof. 18.The method of claim 13 or 17, wherein the compound of formula I is oneof Compounds 1-8:


19. The method of claim 13 or 17, wherein the disorder is cancer, solidtumor, colon cancer, breast cancer, bone, brain and others (e.g.,osteosarcoma, neuroblastoma, colon adenocarcinoma the like.
 20. Themethod of claim 13 or 17, wherein the disorder is an angiogenesisdisorder.
 21. The method of claim 13 or 17, wherein the disorder issolid tumor.
 22. The method of claim 13 or 17, wherein the subject is amammal.
 23. The method of claim 13 or 17 wherein the subject is aprimate or human.
 24. The method of claim 13 or 17, wherein theeffective amount of the compound of formula I ranges from about 0.005μg/kg to about 200 mg/kg.
 25. The method of claim 24, wherein theeffective amount of the compound of formula I ranges from about 0.1mg/kg to about 200 mg/kg.
 26. The method of claim 25, wherein theeffective amount of compound of formula I ranges from about 10 mg/kg to100 mg/kg.
 27. The method of claim 13 or 17, wherein the effectiveamount of the compound of formula I ranges from about 1.0 pM to about500 nM
 28. The method of claim 13 or 17, wherein the compound of formulaI is administered intravenously, intramuscularly, subcutaneously,intracerebroventricularly, orally or topically.
 29. The method of claim13 or 17, wherein the compound of formula I is administered alone or incombination with one or more other therapeutics.
 30. The method of claim29, wherein the additional therapeutic agent is an anti-cancer agent,chemotherapeutic agent, an anti-angiogenesis agent, cytotoxic agent, oran anti-proliferation agent.
 31. A method of treating cancer or tumors,comprising administering to said subject in need thereof, an effectiveamount of any of Compounds 1-8, and pharmaceutically acceptable saltsthereof:


32. A method of treating an histone deacetylase (HDAC) mediated diseaseor disorder, comprising administering to said subject in need thereof,an effective amount of any of Compounds 1-8, and pharmaceuticallyacceptable salts thereof:


33. A method of treating cutaneous T-cell lymphoma, comprisingadministering to said subject in need thereof, an effective amount ofany of Compounds 1-8, and pharmaceutically acceptable salts thereof:


34. A method for treating memory loss, inducing neurogenesis, enhancingmemory retention, enhancing memory formation, increasing synapticpotential or transmission, or increasing long term potentiation (LTP) ina subject, comprising administering to said subject in need thereof, aneffective amount of a compound of formula (I), or pharmaceuticallyacceptable salts thereof:

wherein: each R is independently H or optionally substituted alkyl; eachR¹ is independently H, or optionally substituted alkyl; each R² isindependently H, optionally substituted alkyl, or C(O)R: each R³ isindependently H, optionally substituted alkyl, C(O)OR, or C(O)NRR; eachR⁴ is independently H, optionally substituted alkyl, C(O)OR, or C(O)NRR;and pharmaceutically acceptable salts, solvate, or hydrate thereof. 35.A method for treating diseases and disorders associated with stem cellfate and that are affected by cell differentiation, dedifferentiation ortransdifferentiation in a subject, comprising administering to saidsubject in need thereof, an effective amount of a compound of formula(I), or pharmaceutically acceptable salts thereof:

wherein: each R is independently H or optionally substituted alkyl; eachR¹ is independently H, or optionally substituted alkyl; each R² isindependently H, optionally substituted alkyl, or C(O)R; each R³ isindependently H, optionally substituted alkyl, C(O)OR, or C(O)NRR; eachR⁴ is independently H, optionally substituted alkyl, C(O)OR, or C(O)NRR;and pharmaceutically acceptable salts, solvate, or hydrate thereof.